Divergence of Anti-Angiogenic Activity and Hepatotoxicity of Different Stereoisomers of Itraconazole
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Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Divergence of Anti-angiogenic Activity and Hepatotoxicity of Different Stereoisomers of Itraconazole Joong Sup Shim1,2,3, Ruo-Jing Li1,3, Namandje N. Bumpus1,4, Sarah A. Head1, Kalyan Kumar1, Eun Ju Yang2, Junfang Lv2, Wei Shi1,5, Jun O. Liu1,6 1Department of Pharmacology and Molecular Sciences, 4Department of Medicine and 6Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA 2Faculty of Health Sciences, University of Macau, Taipa, Macau SAR, China 5Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA 3These authors contributed equally to this study. Correspondence to: Jun O. Liu, Ph.D, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N Wolfe St, Hunterian Building 516, Baltimore, MD 21205 (e-mail: [email protected]) Running title: Distinct activity and toxicity of itraconazole stereoisomers Keywords: itraconazole, stereoisomers, drug repositioning, angiogenesis, hepatotoxicity Financial support: This work was supported in part by R01CA184103, the Flight Attendant Medical Research Institute (J. O. L.), the Johns Hopkins Institute for Clinical and Translational Research (ICTR) which is funded in part by Grant Number UL1 TR 001079 and the PhRMA foundation fellowship in Pharmacology/Toxicology (S. A. H.). Conflict of interest: Patents covering itraconazole and its stereoisomers as angiogenesis and hedgehog pathway inhibitors have been licensed from Johns Hopkins to Accelas Pharmaceuticals, of which J.O.L. is a cofounder and owns equity. The potential conflict of 1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. interest is being managed by Johns Hopkins University. No potential conflicts of interest were disclosed by the other authors. Word count: 6059 (excluding title page, references and figures/tables) Total number of figures and tables: 6 2 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance Itraconazole, a known antifungal drug, was found to possess anti-angiogenic and anti-hedgehog activity. However, its clinical development has been limited in part by its hepatotoxicity. We determined the antiangiogenic activity and hepatotoxicity of each of the four individual cis- stereoisomers of itraconazole. We found that one particular stereoisomer, the 2S4R2’S stereoisomer, designated IT-C, has more potent antiangiogenic and anticancer activity with less hepatotoxicity, both in vitro and in animal models, in comparison with the racemic mixture of itraconazole. These results revealed for the first time that stereochemistry of itraconazole has differential effects on its anti-angiogenic activity and hepatotoxicity, suggesting that IT-C is likely to be superior to itraconazole for future clinical development as an anticancer and anti- angiogenic drug. 3 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: Itraconazole is a triazole antifungal drug that has recently been found to inhibit angiogenesis. Itraconazole is a relatively well-tolerated drug but shows hepatotoxicity in a small subset of patients. Itraconazole contains three chiral centers and the commercial itraconazole is composed of four cis-stereoisomers (named IT-A, IT-B, IT-C, and IT-D). We sought to determine whether the stereoisomers of itraconazole might differ in their antiangiogenic activity and hepatotoxicity. Experimental Design: We assessed in vitro antiangiogenic activity of itraconazole and each stereoisomer using human umbilical vein endothelial cell (HUVEC) proliferation and tube formation assays. We also determined their hepatotoxicity using primary human hepatocytes in vitro and a mouse model in vivo. Mouse Matrigel plug and tumor xenograft models were used to evaluate in vivo antiangiogenic and antitumor activities of the stereoisomers. Results: Of the four stereoisomers contained in commercial itraconazole, we found that IT-A (2S,4R,2’R) and IT-C (2S,4R,2’S) were more potent for inhibition of angiogenesis than IT-B (2R,4S,2’R) and IT-D (2R,4S,2’S). Interestingly, IT-A and IT-B were more hepatotoxic than IT- C and IT-D. In mouse models, IT-C showed more potent antiangiogenic/antitumor activity with lower hepatotoxicity compared to itraconazole and IT-A. Conclusions: These results demonstrate the segregation of influence of stereochemistry at different positions of itraconazole on its antiangiogenic activity and hepatotoxicity, with the 2 and 4 positions affecting the former and the 2’ position affecting the latter. They also suggest that IT-C may be superior to the racemic mixture of itraconazole as an anticancer drug candidate due to its lower hepatotoxicity and improved antiangiogenic activity. 4 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Angiogenesis, the formation of new blood vessels from pre-existing vasculature, has been shown to play a critical role in both normal physiological and pathological processes. It is essential during development and wound healing, and is tightly regulated by endogenous pro- and anti- angiogenic factors. It has also been implicated in a number of diseases including cancer, rheumatoid arthritis and macular degeneration (1-3). Since the angiogenesis hypothesis was first proposed by Judah Folkman in 1971 (4), a number of angiogenesis inhibitors have been discovered and developed into anti-angiogenic drugs (5-7). A monoclonal antibody against vascular endothelial growth factor (VEGF), bevacizumab, was first approved by the US-Food and Drug Administration (FDA) in 2004 for the treatment of metastatic colon cancer in combination with standard chemotherapy (8). Subsequently, several small molecule angiogenesis inhibitors have been entered the clinic, including sorafenib (Nexavar®), sunitinib (Sutent®) and pazopanib (Votrient®). However, most of these small molecule antiangiogenic drugs are kinase inhibitors that lack specificity or lead to a high frequency of drug resistance (9-11), necessitating the development of small molecule angiogenesis inhibitors with novel mechanisms of action. In an effort to accelerate drug discovery and development, we assembled the Johns Hopkins Drug Library over a decade ago and screened it for new anti-angiogenic activity among existing drugs using a proliferation assay with primary human umbilical vein endothelial cells. Among the most interesting hits was the antifungal drug itraconazole (12). Interestingly, itraconazole was also found to be an inhibitor of the hedgehog signaling pathway in a separate screen, rendering itraconazole a novel anticancer drug candidate capable of inhibiting the growth of both tumor vasculature and tumor cells themselves (13). A series of tests of itraconazole in preclinical angiogenesis and cancer models confirmed the anti-angiogenic and anticancer activity of itraconazole in vivo (12-14). Based on these promising preclinical results, itraconazole entered multiple Phase II human clinical studies for treating different types of cancer. (http://www.clinicaltrials.gov/). Positive clinical results have been reported for advanced lung cancer, metastatic prostate cancer and basal cell carcinoma (15-17). Moreover, itraconazole was found to increase progression or overall survival among late-stage ovarian, triple-negative breast and metastatic pancreatic cancer patients upon retrospective analysis of previous clinical data 5 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 22, 2016; DOI: 10.1158/1078-0432.CCR-15-1888 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. (18-21). Together, these encouraging results strongly suggest that itraconazole is a promising new anticancer drug lead. Although the precise mechanism for the antiangiogenic and anti-hedgehog activities remains unknown, preliminary evidence suggests that the mode of action of itraconazole is distinct from any other known inhibitors of angiogenesis or the hedgehog signaling pathway. Early on, we ruled out the cholesterol biosynthetic enzyme, lanosterol 14α-demethylase, which mediates the antifungal activity of itraconazole, as the relevant target for its antiangiogenic activity (12). Subsequently, we serendipitously